The dopamine D2-like receptors are targets for antipsychotic drugs used in the treatment of schizophrenia and other psychotic illness. The goal of this project is to understand the structural bases of agonist and antagonist binding to the dopamine D2 and D4 receptors. Considerable evidence indicates that the binding sites of these receptors are formed among the membrane-spanning segments. These sites---are within the plane of the membrane-bilayer, yet accessible to charged, water-soluble agonists like dopamine. Therefore, a waterfilled crevice open to the extracellular medium must exist within the membrane-spanning domain of the protein, with its surface formed by amino acid residues contributed by more than one membrane-spanning segment. The goal of this project is to identify the residues which form this surface. Residues in putative membrane-spanning segments will be mutated to cysteine, one at a time. The cysteine- substitution mutants will be expressed in HEK 293 cells, and antagonist binding to the mutant receptors will be examined in intact cells. If binding is near-normal, the mutant receptor will be probed with small, charged, hydrophilic, sulfhydryl-specific reagents, which are derivatives of methanethiosulfonate. These reagents covalently attach a charged group to the engineered cysteine. In membrane-spanning segments these highly polar reagents will react only with side chains of residues that are accessible from the binding-site crevice and not with residues that face into the hydrophobic core of the protein or into the lipid ,bilayer. Thus, if binding to a mutant receptor is irreversIbly blocked by the sulfhydryl- specific reagents, and If D2 antagonists and agonists can prevent this reaction, we infer that the side chain of the corresponding wild-type residue forms part of the surface of the binding-site crevice. Since antagonist binding to wild-type D2 receptor is blocked irreversibly by the sulfhydryl reagents, the sensitive cysteine had to be identified before applying this mapping approach. Cys118 in the third membrane-spanning segment (TMS III) was found to be responsible for the sensitivity of wild- type receptor to hydrophilic sulfhydryl reagents. Starting with receptor in which Cys118 was replaced by serine, several other residues in TMS III which also line the binding site have been identified with the cysteine- substitution approach. This approach will be applied to all residues in the membrane-spanning segments of the, D2 receptor, thereby mapping the entire surface of the binding site crevice. The approach will also be applied to the residues in the D4 receptor that align with the accessible residues in the D2 receptor binding site crevice. Comparison of the residues lining the binding, site crevice in these two receptors will provide a basis for understanding the binding of dopamine agonists and antagonists and of the therapeutically important differences in their binding specificities in particular the higher affinity of the D4 receptor for clozapine.